Effects of Follicular Fluid on Physiological Characteristics and Differentiation of Fallopian Tube Epithelial Cells Implicating for Ovarian Cancer Pathogenesis.

The fallopian tube (FT) is an important reproductive organ in females. Ample evidence suggests that the distal end of FT is the original site of high-grade serous ovarian carcinoma (HGSC). FT may suffer from repeated injury and repair stimulated by follicular fluid (FF); however, this hypothesis has not been examined. In fact, the molecular mechanism of homeostasis, differentiation, and the transformation of fallopian tube epithelial cells (FTECs) resulting from the stimulation of FF are still enigmatic. In this study, we examined the effects of FF along with factors present in the FF on a variety of FTEC models, including primary cell culture, ALI (air-liquid interface) culture, and 3D organ spheroid culture. We found that FF plays a similar role to estrogen in promoting cell differentiation and organoid formation. Moreover, FF significantly promotes cell proliferation and induces cell injury and apoptosis in high concentrations. These observations may help us to investigate the mechanisms of the initiation of HGSC.

New understanding of Angelica sinensis polysaccharide improving fatty liver: The dual inhibition of lipid synthesis and CD36-mediated lipid uptake and the regulation of alcohol metabolism.

Angelica sinensis polysaccharide (ASP) has presented increasingly recognized lipid regulation and antioxidant abilities. However, there is little direct evidence to explain why ASP possesses the observed lipid-lowering and anti-oxidation effects. In vivo and in vitro models of alcoholic fatty liver disease (AFLD) were established to examine the direct effect of ASP on hepatic fat accumulation. Our results showed that the lipid-lowering effect of ASP might result from the dual inhibition of lipid synthesis and CD36-mediated lipid uptake. The antioxidation of ASP might be attributed to the reversal of alcohol metabolic pathways from CYP2E1 catalysis to ADH catalysis. Taken together, the study demonstrated the direct role of ASP in lipid metabolism for the first time and revealed the underlying mechanism of reducing ROS, providing an available strategy for ASP as a potential agent to treat AFLD.

Macrophage elastase derived from adventitial macrophages modulates aortic remodeling.

Abdominal aortic aneurysm (AAA) is pathologically characterized by intimal atherosclerosis, disruption and attenuation of the elastic media, and adventitial inflammatory infiltrates. Although all these pathological events are possibly involved in the pathogenesis of AAA, the functional roles contributed by adventitial inflammatory macrophages have not been fully documented. Recent studies have revealed that increased expression of matrix metalloproteinase-12 (MMP-12) derived from macrophages may be particularly important in the pathogenesis of both atherosclerosis and AAA. In the current study, we developed a carrageenan-induced abdominal aortic adventitial inflammatory model in hypercholesterolemic rabbits and evaluated the effect of adventitial macrophage accumulation on the aortic remodeling with special reference to the influence of increased expression of MMP-12. To accomplish this, we compared the carrageenan-induced aortic lesions of transgenic (Tg) rabbits that expressed high levels of MMP-12 in the macrophage lineage to those of non-Tg rabbits. We found that the aortic medial and adventitial lesions of Tg rabbits were greater in degree than those of non-Tg rabbits, with the increased infiltration of macrophages and prominent destruction of elastic lamellae accompanied by the frequent appearance of dilated lesions, while the intimal lesions were slightly increased. Enhanced aortic lesions in Tg rabbits were focally associated with increased dilation of the aortic lumens. RT-PCR and Western blotting revealed high levels of MMP-12 in the lesions of Tg rabbits that were accompanied by elevated levels of MMP-2 and -3, which was caused by increased number of macrophages. Our results suggest that adventitial inflammation constitutes a major stimulus to aortic remodeling and increased expression of MMP-12 secreted from adventitial macrophages plays an important role in the pathogenesis of vascular diseases such as AAA.

Reductively modified albumin attenuates DSS-Induced mouse colitis through rebalancing systemic redox state.

Albumin (Alb) is the most abundant plasma protein with multiple biological functions, including antioxidative property through its thiol activity. Given that inflammatory bowel disease is associated with a decreased level of Alb and an increased level of Alb oxidation, we asked whether Alb could have a therapeutic effect on colitis. Here we tested this possibility. Bovine serum albumin (BSA) was reductively modified with dithiothreitol (DTT) and administrated via gavage or intraperitoneal injection. Dextran sulfate sodium (DSS)-induced mice colitis was associated with massive oxidative stress, as indicated by the elevated sulfenic acid formation in blood, colon tissues, and feces. Treatment of mice with the reductively modified albumin (r-Alb) attenuated the oxidative stress and reduced local inflammation and tissue injury. These effects of r-Alb were only partially achieved by unmodified Alb and wholly lost after blocking the -SH groups with maleimide. In cultured colon epithelial cells, r-Alb prevented DSS- and H2O2-induced ROS elevation and barrier dysfunction, preceded by inhibition of sulfenic acid formation and P38 activation. Further analysis revealed that Alb was susceptible to H2O2-induced oxidation, and it detoxified H2O2 in a -SH group-dependent way. Moreover, Alb reacted with GSH/GSSG via thiol-disulfide exchange and reciprocally regulated the availability of -SH groups. Collectively, our study shows that r-Alb effectively attenuates DSS colitis via -SH group-mediated antioxidative action. Given that the oxidative stress underlies many life-threatening diseases, r-Alb, functioning as a potent antioxidant, could have a wide range of applications.

Possible mechanisms by which silkworm faeces extract ameliorates adenine-induced renal anaemia in rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Silkworm faeces are the dry faeces of the insect Bombyx mori (Linnaeus) and have historically been used in traditional Chinese medicine to treat blood deficiency and rheumatic pain. Silkworm faeces extract (SFE) is derived from silkworm faeces. AIM OF THE STUDY: Clinical observations of patients in the Department of Nephrology have shown that SFE effectively improves renal anaemia. However, the molecular mechanism remains unclear. This article mainly explores the regulatory effects of SFE on erythropoietin (EPO) and hepcidin to identify the molecular mechanism of SFE. MATERIALS AND METHODS: A rat model of renal anaemia was established by feeding rats food containing 0.75% adenine. SFE was orally administered to the rats, while recombinant human erythropoietin (rhEPO) was used as a positive control drug. Haematological parameters and inflammation levels were compared between rats from each group, and pathological kidney sections from each rat were observed. The serum EPO and hepcidin levels were detected using enzyme-linked immunosorbent assay (ELISA) kits, while Western blot analyses were performed to detect the levels of proteins involved in the EPO-related hypoxia-inducible factor 2α (HIF-2α)/prolyl hydroxylase 2 (PHD2) signalling pathway and hepcidin-related BMP6/SMAD4 and interleukin-6 (IL-6)/STAT3 signalling pathways. RESULTS: SFE significantly ameliorated haematological parameters, renal function, and inflammation levels in the rats. A mechanistic study showed that SFE promoted EPO expression by upregulating HIF-2α expression and inhibiting the expression of NF-κB and GATA2 both in vivo and in vitro. In particular, SFE inhibited PHD2 expression, resulting in a decrease in the enzymatic reaction of HIF-2α to increase EPO expression. Furthermore, SFE inhibited hepcidin expression by blocking the BMP6/SMAD4 and IL-6/STAT3 pathways. CONCLUSIONS: SFE regulated iron metabolism by inhibiting hepcidin and simultaneously promoted EPO synthesis to improve renal anaemia in rats.

Apolipoprotein CIII Deficiency Protects Against Atherosclerosis in Knockout Rabbits.

OBJECTIVE: Apo (apolipoprotein) CIII mediates the metabolism of triglyceride (TG)-rich lipoproteins. High levels of plasma apoCIII are positively correlated with the plasma TG levels and increase the cardiovascular risk. However, whether apoCIII is directly involved in the development of atherosclerosis has not been fully elucidated. Approach and Results: To examine the possible roles of apoCIII in lipoprotein metabolism and atherosclerosis, we generated apoCIII KO (knockout) rabbits using ZFN (zinc finger nuclease) technique. On a normal standard diet, apoCIII KO rabbits exhibited significantly lower plasma levels of TG than those of WT (wild type) rabbits while total cholesterol and HDL (high-density lipoprotein) cholesterol levels were unchanged. Analysis of lipoproteins isolated by sequential ultracentrifugation revealed that reduced plasma TG levels in KO rabbits were accompanied by prominent reduction of VLDLs (very-low-density lipoproteins) and IDLs (intermediate-density lipoproteins). In addition, KO rabbits showed faster TG clearance rate after intravenous fat load than WT rabbits. On a cholesterol-rich diet, KO rabbits exhibited constantly and significantly lower levels of plasma total cholesterol and TG than WT rabbits, which was caused by a remarkable reduction of ß-VLDLs-the major atherogenic lipoproteins. ß-VLDLs of KO rabbits showed higher uptake by cultured hepatocytes and were cleared faster from the circulation than ß-VLDLs isolated from WT rabbits. Both aortic and coronary atherosclerosis was significantly reduced in KO rabbits compared with WT rabbits. CONCLUSIONS: These results indicate that apoCIII deficiency facilitates TG-rich lipoprotein catabolism, and therapeutic inhibition of apoCIII expression may become a novel means not only for the treatment of hyperlipidemia but also for atherosclerosis.

Dendrobium officinale Polysaccharide Protected CCl4-Induced Liver Fibrosis Through Intestinal Homeostasis and the LPS-TLR4-NF-κB Signaling Pathway.

We explored the therapeutic effects of Dendrobium officinale polysaccharide (DOP) on CCl4-induced liver fibrosis with respect to the intestinal hepatic axis using a rat model. Histopathological staining results showed that DOP alleviated extensive fibrous tissue proliferation in interstitium and lessened intestinal mucosal damage. Western blot and PCR results showed that DOP maintained intestinal balance by upregulating the expression of tight junction proteins such as occludin, claudin-1, ZO-1, and Bcl-2 proteins while downregulating the expression of Bax and caspase-3 proteins in the intestine. The transepithelial electrical resistance (TEER) value of the LPS-induced Caco-2 monolayer cell model was increased after DOP administration. These illustrated that DOP can protect the intestinal mucosal barrier function. DOP also inhibited activation of the LPS-TLR4-NF-κB signaling pathway to reduce the contents of inflammatory factors TGF-ß and TNF-α, increased the expression of anti-inflammatory factor IL-10, and significantly decreased α-SMA and collagen I expression. These results indicated that DOP maintained intestinal homeostasis by enhancing tight junctions between intestinal cells and reducing apoptosis, thereby inhibiting activation of the LPS-TLR4-NF-κB signaling pathway to protect against liver fibrosis.

Hydrogen Sulfide Mediates Tumor Cell Resistance to Thioredoxin Inhibitor.

Thioredoxin (Trx) is a pro-oncogenic molecule that underlies tumor initiation, progression and chemo-resistance. PX-12, a Trx inhibitor, has been used to treat certain tumors. Currently, factors predicting tumor sensitivity to PX-12 are unclear. Given that hydrogen sulfide (H2S), a gaseous bio-mediator, promotes Trx activity, we speculated that it might affect tumor response to PX-12. Here, we tested this possibility. Exposure of several different types of tumor cells to PX-12 caused cell death, which was reversely correlated with the levels of H2S-synthesizing enzyme CSE and endogenous H2S. Inhibition of CSE sensitized tumor cells to PX-12, whereas addition of exogenous H2S elevated PX-12 resistance. Further experiments showed that H2S abolished PX-12-mediated inhibition on Trx. Mechanistic analyses revealed that H2S stimulated Trx activity. It promoted Trx from the oxidized to the reduced state. In addition, H2S directly cleaved the disulfide bond in PX-12, causing PX-12 deactivation. Additional studies found that, besides Trx, PX-12 also interacted with the thiol residues of other proteins. Intriguingly, H2S-mediated cell resistance to PX-12 could also be achieved through promotion of the thiol activity of these proteins. Addition of H2S-modified protein into culture significantly enhanced cell resistance to PX-12, whereas blockade of extracellular sulfhydryl residues sensitized cells to PX-12. Collectively, our study revealed that H2S mediated tumor cell resistance to PX-12 through multiple mechanisms involving induction of thiol activity in multiple proteins and direct inactivation of PX-12. H2S could be used to predict tumor response to PX-12 and could be targeted to enhance the therapeutic efficacy of PX-12.

Connexin43 Is Required for the Effective Activation of Spleen Cells and Immunoglobulin Production.

Gap junctions (Gjs), formed by specific protein termed connexins (Cxs), regulate many important cellular processes in cellular immunity. However, little is known about their effects on humoral immunity. Here we tested whether and how Gj protein connexin43 (Cx43) affected antibody production in spleen cells. Detection of IgG in mouse tissues and serum revealed that wild-type (Cx43+/+) mouse had a significantly higher level of IgG than Cx43 heterozygous (Cx43+/-) mouse. Consistently, spleen cells from Cx43+/+ mouse produced more IgG under both basal and lipopolysaccharide (LPS)-stimulated conditions. Further analysis showed that LPS induced a more dramatic activation of ERK and cell proliferation in Cx43+/+ spleen cells, which was associated with a higher pro-oxidative state, as indicated by the increased NADPH oxidase 2 (NOX2), TXNIP, p38 activation and protein carbonylation. In support of a role of the oxidative state in the control of lymphocyte activation, exposure of spleen cells to exogenous superoxide induced Cx43 expression, p38 activation and IgG production. On the contrary, inhibition of NOX attenuated the effects of LPS. Collectively, our study characterized Cx43 as a novel molecule involved in the control of spleen cell activation and IgG production. Targeting Cx43 could be developed to treat certain antibody-related immune diseases.

Connexin43 Contributes to Inflammasome Activation and Lipopolysaccharide-Initiated Acute Renal Injury via Modulation of Intracellular Oxidative Status.

Aims: Inflammasome activation plays a pivotal role in many inflammatory diseases. Given that connexin (Cx) channels regulate numerous cellular events leading to inflammasome activation, we determined whether and how connexin affected inflammasome activation and inflammatory cell injury. Results: Exposure of mouse peritoneal macrophages (PMs) to lipopolysaccharide (LPS) plus ATP caused NLRP3 inflammasome activation, together with an increased connexin43 (Cx43). Inhibition of Cx43 blunted inflammasome activation. Consistently, PMs from the Cx43 heterozygous mouse (Cx43+/-) exhibited weak inflammasome activation, in comparison with those from the Cx43+/+ mouse. Further analysis revealed that inflammasome activation was preceded by an increased reactive oxygen species (ROS) production, nicotinamide adenine dinucleotide phosphate hydrogen (NADPH) oxidase 2 (NOX2), protein carbonylation, and mitogen-activated protein kinase (MAPK) activation. Suppression of ROS with antioxidant, downregulation of NOX2 with small interfering RNA (siRNA), or inhibition of NADPH oxidase or MAPKs with inhibitors blocked Cx43 elevation and inflammasome activation. Intriguingly, suppression of Cx43 also blunted NOX2 expression, protein carbonylation, p38 phosphorylation, and inflammasome activation. In a model of acute renal injury induced by LPS, the Cx43+/- mouse exhibited a significantly lower level of blood interleukin-1ß (IL-1ß), blood urea nitrogen, and urinary protein, together with milder renal pathological changes and renal expression of NLRP3 and NOX4, as compared with the Cx43+/+ mouse. Moreover, inhibition of gap junctions suppressed IL-1ß- and tumor necrosis factor-α-induced expression of NOX4 in glomerular podocytes and tubular epithelial cells. Innovation and Conclusion: Our study indicates that Cx43 contributes to inflammasome activation and the progression of renal inflammatory cell injury through modulation of intracellular redox status. Cx43 could be a novel target for the treatment of certain inflammatory diseases.

Hydrogen sulfide donor NaHS alters antibody structure and function via sulfhydration.

Hydrogen sulfide (H2S) has emerged as an important biological mediator with numerous pathophysiological roles. One of the well-documented actions of H2S is to inhibit immunity, especially cellular immunity. Currently, limited information is available regarding its effects on humoral immunity. Given that H2S has reducing activity and that the effector molecules in humoral immunity, such as antibody and complement, contain abundant disulfide bonds that are indispensable for their functions, we speculated that H2S might regulate antibody activity via modification of disulfide bonds. Here we addressed this possibility. Exposure of antibodies to H2S donors resulted in cleavage of the disulfide bonds between the heavy and light chains of antibodies, which was associated with antibody sulfhydration. Further analysis revealed that H2S-treated antibodies exhibited a marked reduction in antigen binding ability. It potently prevented the antibody-mediated agglutination of red blood cells and interrupted aggregation of antibody-coated microspheres. H2S also greatly inhibited antibody-induced and complement-mediated cell lysis in glomerular mesangial cells, as well as anti-CD95 IgM antibody-initiated cell apoptosis in Jurkat cells. Moreover, it significantly suppressed the alternative complement activation pathway. Collectively, our results revealed, for the first time, that pharmacologic levels of H2S inhibit humoral immune responses via direct sulfhydration of the effector molecules. Our study thus provides novel mechanistic insights into the immunoregulatory actions of H2S and suggests that H2S may have potential to treat certain humoral immune diseases.

Pharmacological levels of hydrogen sulfide inhibit oxidative cell injury through regulating the redox state of thioredoxin.

Hydrogen sulfide (H2S) is a gaseous mediator with multifaceted biological activities. It has anti-inflammatory and anti-oxidative effects. Currently, the mechanisms are not fully understood. Given that Trx/ASK1/P38 signaling pathway mediates many oxidative cell responses, we tested whether and how H2S affected this pathway. Exposure of podocytes to Adriamycin (ADR), an antitumor drug, led to a P38-mediated oxidative cell injury, as evidenced by the increased protein carbonylation, oxidative activation of P38, and prevention of the cell death by antioxidants, NADPH oxidase inhibitor and P38 inhibitor. In the presence of H2S donor NaHS, however, the podocyte injury was largely prevented. NaHS also significantly prevented cell death elicited by H2O2, menadione, and thioredoxin (Trx) inhibitors. These effects of H2S were also associated with a potent inhibition of P38. Further analysis revealed that H2S did not affect the protein level of TXNIP and Trx, two pivotal regulators of ASK1/P38 activation, but it promoted the dissociation of Trx from TXNIP. Moreover, it disrupted the H2O2-initiated polymerization of Trx and converted Trx from the oxidized to the reduced form. In HepG2 cells, inhibition of H2S-producing enzyme cystathionine γ-lyase (CSE) increased Trx oxidation, promoted Trx binding to TXNIP and exaggerated cell injury caused by Trx inhibition. Collectively, our results indicate that H2S exerted its antioxidative effects through the regulation of the redox state of Trx and interference with Trx/ASK1/P38 signaling pathway. Given the importance of the pathway in the mediation of multiple oxidative cell responses, our study thus provides novel mechanistic insight into the action of H2S.

A sensitive and rapid radiolabelling method for the in vivo pharmacokinetic study of lentinan.

The aim of this study is to establish a rapid and sensitive method for detecting lentinan (LNT) in biosamples and to evaluate the pharmacokinetics of LNT in mice and rats. A diethylenetriaminepentaacetic acid (DTPA) derivative of LNT (DTPA-LNT) was synthesized first to allow labelling with 99m-technetium (99mTc). After purification and identification, 99mTc-DTPA-LNT was intravenously administered to mice (2 mg kg-1) and rats at different doses (0.5, 2 and 8 mg kg-1). The results showed that the 99mTc-labelling method was suitable for the quantification of the LNT concentration in biological samples, with satisfactory linearity (r2 > 0.998), precision (<7%), accuracy (95.01-104.51%) and total recovery (∼90%). The blood concentration-time profiles of 99mTc-DTPA-LNT were consistent with the two-compartment model and showed a rapid distribution phase and a slow elimination, and no significant difference in the blood level of LNT was found among the tested doses (0.5, 2 and 8 mg kg-1). LNT was predominantly incorporated into the liver and spleen, and there was a small amount of aggregation in the bile, kidneys, lungs and stomach. Approximately 40% of the administered radioactivity was detected in urine and faeces within 24 h post-dosing. In addition, SPECT imaging of 99mTc-DTPA-LNT was performed to visually reveal the pharmacokinetic characteristics of LNT. These findings provide a reference for further study and for use of LNT and other ß-glucans.

Induction of inactive TGF-ß1 monomer formation by hydrogen sulfide contributes to its suppressive effects on Ang II- and TGF-ß1-induced EMT in renal tubular epithelial cells.

Hydrogen sulfide (H2S), an endogenous gas mediator with multifaced biological functions, has been shown to be effective in the prevention and treatment of renal sclerosis in several models of chronic renal diseases. The mechanisms involved are still unclear. Given that Ang II- and TGF-ß-induced renal tubular epithelial-mesenchymal transition (EMT) is a pivotal cellular event leading to renal sclerosis, we examined whether and how H2S intervened the processes of EMT. Ang II stimulated EMT in renal tubular epithelial cells, as indicated by the increased level of α-smooth muscle actin and a decreased level of E-cadherin. This effect of Ang II was blocked by a TGF-ß receptor kinase inhibitor, indicative of a mediating role of TGF-ß. Consistently, Ang II stimulated TGF-ß activation and addition of the exogenous TGF-ß1 also induced EMT. In the presence of H2S donor NaHS, the EMT-promoting actions of Ang II and TGF-ß1 were abolished, which was associated with a reduced TGF-ß activity. Further analysis using a human recombinant active TGF-ß1 revealed that H2S cleaved the disulfide bond in the dimeric active TGF-ß1 and promoted the formation of inactive TGF-ß1 monomer. Collectively, these results indicate that H2S counteracted Ang II- and TGF-ß1-induced EMT through mechanisms involving direct inactivation of TGF-ß1. Our study thus provides novel mechanistic insight into the anti-fibrotic actions of H2S and suggest that H2S could be used to treat renal sclerotic diseases.

Constitutive activated STAT3 is an essential regulator and therapeutic target in esophageal squamous cell carcinoma.

Esophageal carcinoma is among the most common cancers worldwide and a leading cause of cancer death [1]. Large numbers of studies indicated that chronic inflammation is closely associated with its development [21, 25]. Furthermore, the JAK/STAT pathway, which plays a critical role in inflammation and immunity, has been implied in a number of malignancies [11]. It has been shown that targeting the pathway affected the growth, apoptosis, and metastasis of cultured esophageal squamous cell carcinoma cells [26]. We found in the present study that STAT3 is constitutively activated in a subgroup of esophageal squamous cell carcinoma cell lines and primary tumors. Altered expressions of STAT3 target genes were found in these tumors by using RNAseq and qPCR analysis. Cytokines that activate STAT3 affected the expression of STAT3 target genes and promoted the growth of the ESCC cells, which could be blocked by STAT3 inhibitor and specific siRNA. Inhibition of STAT3 also suppressed the growth and colony formation, and induced apoptosis in the esophageal squamous cell carcinoma cells containing constitutively activated STAT3. Furthermore, the STAT3 inhibitor effectively blocked the growth of patient-derived tumor xenografts that harbored phosphorylated STAT3, but acted less effective on the xenografts derived from primary tumors that contained low levels of activated STAT3. These results indicated that activated STAT3 plays a critical role in the survival and growth of a subgroup of esophageal squamous cell carcinoma, and may serve as a target for precision therapeutic intervention.

A method for establishing a patient-derived xenograft model to explore new therapeutic strategies for esophageal squamous cell carcinoma.

Esophageal squamous cell carcinoma (ESCC) is the predominant histological type of esophageal carcinoma in China. The overall 5-year survival rate of ESCC patients is in the low range of 15-25%. One important reason for the poor prognosis is that the underlying molecular mechanisms are unclear. Furthermore, the development of effective therapeutic strategies to improve patient outcome is needed. Animal models can be beneficial to analyze the molecular mechanisms as well as specific clinical therapeutic strategies for esophageal cancer. In recent years, patient-derived xenografts (PDXs) have been widely used in numerous types of cancers to investigate the basic mechanisms and to conduct preclinical research. Accumulating evidence indicates that the PDX model is an important tool for basic and clinical research. Herein, we successfully established 14 ESCC PDXs. These PDX models preserved the patient pathological characteristics and effectively reflected the patient biological heterogeneity. Cancers exhibit diverse growth rates and tumor texture, even more, they have different signaling pathways. The PDX model is a superior strategy for understanding the underlying molecular mechanisms of ESCC and for screening new therapeutic strategies for ESCC patients.